Systems and methods for characterizing kidney disease

ABSTRACT

The present invention relates to methods of diagnosing, predicting and monitoring kidney disorders. In particular, the present invention relates to the diagnosis, prediction and monitoring of kidney disorders by detection of cytokines, cytokine-related compounds and chemokines in urine. The present invention further relates to methods and compositions for assessing the efficacy of agents and interventions used to treat kidney disorders.

The present application is a continuation-in-part of U.S. applicationSer. No. 10/903,797, filed Jul. 30, 2004, which is acontinuation-in-part of U.S. application Ser. No. 10/313,807, filed Dec.6, 2002, each of which is incorporated herein by reference in itsentirety. The present application also claims priority from U.S.provisional patent application Ser. Nos. 60/491,900, filed Aug. 1, 2003and 60/499,937, filed Sep. 3, 2003, each of which is herein incorporatedherein by reference in its entirety.

FIELD OF THE INVENTION

The present invention relates to methods of diagnosing, predicting andmonitoring kidney disorders. In particular, the present inventionrelates to the diagnosis, prediction and monitoring of kidney disordersby detection of cytokines, cytokine-related compounds, and chemokines inurine. The present invention further relates to methods and compositionsfor assessing the efficacy of agents and interventions used to treatkidney disorders.

BACKGROUND OF THE INVENTION

The diagnosis of kidney disorders is classically based on the presenceof one or more signs and symptoms. For example, signs and symptoms ofacute renal failure, renal tubular injury, renal cancer orglomerulonephritis may include weight gain, reduced urine output,increased serum creatine concentrations, hypertension, fever, and kidneyenlargement and tenderness. However, the use of these signs and symptomsalone to detect kidney disorders is not adequate. Currently, most kidneydisorders are diagnosed by measuring kidney function, for example byusing biochemical tests such as assays that measure serum creatinine(Cr) concentrations, and by imaging or biopsy.

Presently, renal biopsy remains the most definitive test to specificallydiagnose many kidney disorders. However, renal biopsy has majorlimitations. For example, the biopsy procedure itself has complications,and cannot be performed on a routine or serial basis to monitorprogression of renal disease. In addition, a renal biopsy is invasive,making it uncomfortable, inconvenient and often dangerous for patients.Moreover, accurate interpretation of a renal biopsy demands theexpertise of a pathologist with extensive experience in analyzing thesample for evidence of specific kidney disorders. Hence, renal biopsiesare reserved for those patients demonstrating other clinical and/orlaboratory evidence of a kidney disorders, thus limiting its broaderuse.

Thus, a less invasive method for the diagnosis, prediction andmonitoring of kidney disorder is clearly needed.

SUMMARY OF THE INVENTION

The present invention relates to methods of diagnosing, predicting andmonitoring kidney disorders. In particular, the present inventionrelates to the diagnosis, prediction and monitoring of kidney disordersby detection of cytokines, cytokine-related compounds or chemokines inurine, or in other body fluids, for example, blood, serum, plasma, bile,saliva, or cerebrospinal fluid. The present invention further relates tomethods and compositions for assessing the efficacy of agents andinterventions used to treat kidney disorders.

Accordingly, in some embodiments, the present invention provides amethod for diagnosing disorders of the kidney comprising providing aurine sample from a subject, wherein said subject is suspected of havinga kidney disorder, providing reagents for detection of at least onecompound from the list comprising adiponectin, IGFBP-1, IGFBP-2,IGFBP-6, IL-8, leptin, MCP-1, MIP-1δ, TNF sR1, osteoprotogerin, anduPAR, and detecting the presence of said compound in said urine sampleusing said reagents. In some embodiments, said list of said compoundsfurther comprises IP-10 and Mig. In other embodiments, said detectingthe presence of said compound in said urine sample comprises detectingthe amount of said compound in said urine sample. In other embodiments,said method further comprises providing a sample additive compositioncomprising a high concentration salt buffer, wherein said salt buffer,when mixed with an equal volume of urine and said reagents fordetection, provides a concentration of said salt of 200-600 mM in saidmixture.

In some embodiments, said compound is a full size compound. In otherembodiments said compound is a fragment of said full size compound. Infurther embodiments, said reagents comprise reagents for performing animmunoassay. In preferred embodiments said immunoassay is selected fromthe group comprising an ELISA, radio-immunoassay, automated immunoassay,cytometric bead assay, and immunoprecipitation assay. In otherembodiments, said reagents comprise reagents for performing afluorescently activated cell sorting assay. In some embodiments, thepresent invention further comprises the step of determining a treatmentcourse of action based on said diagnosis of a kidney disorder. In otherembodiments, the present invention further comprises the step ofdetermining the presence or absence of a concurrent infection in saidsubject.

The present invention further provides a method of diagnosing a kidneydisorder, comprising providing a urine sample from a subject, providingreagents for detection of two or more compounds from the list comprisingadiponectin, IGFBP-1, IGFBP-2, IGFBP-6, IL-8, leptin, MCP-1, MIP-1δ, TNFsR1, osteoprotogerin, and uPAR, detecting the presence of said two ormore compounds from said list, and diagnosing a kidney disorder in saidsubject based on the result of said detecting. In some embodiments, saidlist of said compounds further comprises IP-10 and Mig. In otherembodiments, said detecting the presence of said compound in said urinesample comprises detecting the amount of said compound in said urinesample. In some embodiments, said method further comprises the step ofdetermining a treatment course of action based on said diagnosis of akidney disorder. In other embodiments said method further comprises thestep of determining the presence or absence of a concurrent infection insaid subject.

The present invention additionally provides a method for distinguishingacute renal graft rejection from chronic renal graft rejection,comprising providing a urine sample from a subject, wherein said subjectis suspected of having renal graft rejection, and reagents for detectionof at least one cytokine, cytokine-related compound or chemokine, anddetecting the presence of said cytokine, cytokine-related compound orchemokine in said urine sample using said reagents. In some embodiments,said detecting the presence of said cytokine compound in said urinesample comprises detecting the amount of said cytokine compound in saidurine sample.

In further embodiments, the present invention provides a kit, comprisingreagents for the detection of the amount one or more compounds selectedfrom the list comprising adiponectin, IGFBP-1, IGFBP-2, IGFBP-6, IL-8,leptin, MCP-1, MIP-1δ, TNF sR1, osteoprotogerin, and uPAR, instructionsfor using said reagents for detecting the presence of one or more ofsaid compounds, and instructions for using said detecting the presenceof said one or more compounds in said urine sample for diagnosing akidney disorder. In some embodiments, said list of said compoundsfurther comprises IP-10 and Mig. In other embodiments, said detectingthe presence of said compound in said urine sample comprises detectingthe amount of said compound in said urine sample. In furtherembodiments, said kit further comprises a sample additive compositioncomprising a high concentration salt buffer, wherein said salt buffer,when mixed with an equal volume of urine and said reagents fordetection, provides a concentration of said salt of 200-600 mM in saidmixture. In some embodiments, said instructions comprise instructionsrequired by the United States Food and Drug Administration for use in invitro diagnostic products. In still further embodiments, said kitfurther comprises second reagents for determining the presence orabsence of a concurrent infection in said subject, and secondinstructions for using said reagents for determining the presence orabsence of said concurrent infection in said subject.

In some embodiments, the present invention provides a method ofdetecting kidney disease markers, comprising providing a urine samplefrom a subject, wherein said subject is suspected of having acute renalfailure, renal tubular interstitial disease or glomerulonephritis;reagents for detection of a CXCR3 ligand or CCR-5 receptor ligand (e.g.,CCL chemokines); and detecting the presence of said ligand in said urinesample using said reagents. In some embodiments, the method furtherprovides the step of predicting renal failure risk in the subject basedon the result of the detecting. In other embodiments, the method furtherprovides the step of detecting renal failure risk in the subject basedon the result of the detecting. In some embodiments, detecting thepresence of the ligand in the urine sample comprises detecting theamount of the ligand in the urine sample. The present invention is notlimited to the detection of a particular ligand. Any suitable ligand iscontemplated including, but not limited to, IP-10, Mig, I-TAC, MIP-1α,MIP-3α, and MIP-1β. In some embodiments, the ligand is a full lengthligand. In other embodiments, the ligand is a fragment of the fulllength ligand. The present invention is not limited to a particularassay. In some embodiments, the reagents comprise reagents forperforming an immunoassay. For example, any suitable immunoassay iscontemplated including, but not limited to, ELISA, radio-immunoassay,automated immunoassay, cytometric bead assay, and immunoprecipitationassay. In some embodiments, the ELISA is a quantitative ELISA assay. Inother embodiments the assay is a Luminex bead assay. In furtherembodiments, the assay is a protein microarray. In some embodiments, thepresent invention further comprises the step of determining a treatmentcourse of action based on the prediction of acute renal failure, tubularinterstitial disease or glomerulonephritis risk. In some embodiments,the treatment course of action comprises the administration oftherapeutic agents. In some embodiments, the treatment course of actioncomprises a surgical procedure. In additional embodiments the surgicalprocedure comprises renal transplantation. In further embodiments thetreatment course of action comprises dialysis. In some embodiments thedialysis is hemodialysis. In other embodiments the dialysis isperitoneal dialysis. In other embodiments, the treatment course ofaction comprises continued monitoring. In some embodiments, the presentinvention further comprises the step of determining the presence orabsence of a concurrent infection in the subject. In some embodiments,the determining of a concurrent infection comprises determining the bodytemperature of the subject. In other embodiments, the determining of aconcurrent infection comprises the detection of a bacterial infection inthe subject. In still further embodiments, the determining of aconcurrent infection comprises the detection of a viral infection in thesubject.

The present invention further provides a method of diagnosing acuterenal failure, tubular interstitial disease, renal cancer orglomerulonephritis in a subject, comprising providing a urine samplefrom a subject; reagents for detection of a CXCR3 ligand or CCR-5receptor ligand (e.g., CCL chemokines); and detecting the presence ofthe ligand in the urine sample using the reagents; and diagnosing acuterenal failure, tubular interstitial disease, renal cancer orglomerulonephritis in the subject based on the result of the detecting.In some embodiments, detecting the presence of the ligand in the urinesample comprises detecting the amount of the ligand in the urine sample.The present invention is not limited to the detection of a particularligand. Any suitable ligand is contemplated including, but not limitedto, IP-10, Mig, I-TAC, MIP-1α, MIP-3α, and MIP-1β. In some embodiments,the ligand is a full-length ligand. In other embodiments, the ligand isa fragment of the full length ligand. The present invention is notlimited to a particular assay. In some embodiments, the reagentscomprise reagents for performing an immunoassay. For example, anysuitable immunoassay is contemplated including, but not limited to,ELISA, radio-immunoassay, automated immunoassay, cytometric bead assay,and immunoprecipitation assay. In some embodiments, the ELISA is aquantitative ELISA assay. In other embodiments the assay is a Luminexbead assay. In further embodiments, the assay is a protein microarray.In some embodiments, the method further comprises the step ofdetermining a treatment course of action based on the diagnosis of acuterenal failure, tubular interstitial disease or glomerulonephritis. Insome embodiments, the present invention further comprises the step ofdetermining the presence or absence of a concurrent infection in thesubject. In some embodiments, the determining of a concurrent infectioncomprises determining the body temperature of the subject. In otherembodiments, the determining of a concurrent infection comprises thedetection of a bacterial infection in the subject. In still furtherembodiments, the determining of a concurrent infection comprises thedetection of a viral infection in the subject.

The present invention additionally provides a method of determining atreatment course of action, comprising providing a urine sample from asubject, wherein the subject is suspected of having acute renal failure,tubular interstitial disease, renal cancer or glomerulonephritis fordetection of a chemokine; and detecting the amount of the chemokine inthe urine sample using the reagents; and determining a treatment courseof action based on the detecting. In some embodiments, the treatmentcourse of action comprises continued monitoring. In some embodiments,the chemokine comprises a CXCR3 ligand or a CCL chemokine. The presentinvention is not limited to the detection of a particular ligand. Anysuitable ligand is contemplated including, but not limited to, IP-10,Mig, I-TAC, MIP-1α, MIP-3α, and MIP-1β. In some embodiments, the ligandis a full-length ligand. In other embodiments, the ligand is a fragmentof a full-length ligand. The present invention is not limited to aparticular assay. In some embodiments, the reagents comprise reagentsfor performing an immunoassay. For example, any suitable immunoassay iscontemplated including, but not limited to, ELISA, radio-immunoassay,automated immunoassay, cytometric bead assay, and immunoprecipitationassay. In some embodiments, the ELISA is a quantitative ELISA assay. Inother embodiments the assay is a Luminex bead assay. In furtherembodiments, the assay is a protein microarray. In some embodiments, thepresent invention further comprises the step of determining a treatmentcourse of action based on the prediction of acute renal failure, tubularinterstitial disease or glomerulonephritis risk. In some embodiments,the treatment course of action comprises the administration oftherapeutic agents. In some embodiments, the treatment course of actioncomprises a surgical procedure. In additional embodiments the surgicalprocedure comprises renal transplantation. In further embodiments thetreatment course of action comprises dialysis. In some embodiments thedialysis is hemodialysis. In other embodiments the dialysis isperitoneal dialysis. In some embodiments, the present invention furthercomprises the step of determining the presence or absence of aconcurrent infection in the subject. In some embodiments, thedetermining of a concurrent infection comprises determining the bodytemperature of the subject. In other embodiments, the determining of aconcurrent infection comprises the detection of a bacterial infection inthe subject. In still further embodiments, the determining of aconcurrent infection comprises the detection of a viral infection in thesubject.

The present invention also provides a method of screening compounds,comprising providing a sample from a subject, wherein the subject issuspected of having acute renal failure, tubular interstitial disease orglomerulonephritis; an assay with reagents for detection of a CXCR3ligand or CCR-5 receptor ligand (e.g., CCL chemokines); and one or moretest compounds; and administering the test compound to the subject;detecting the amount of the ligand in the sample using the reagents. Thepresent invention is not limited to a particular sample type. Any bodilyfluid including, but not limited to, blood, urine, serum, and lymph maybe utilized. In some preferred embodiments, the sample is a urinesample. In some embodiments, the test compound is a drug. In someembodiments, the method further comprises the step of determining theefficacy of the drug based on the detecting. The present invention isnot limited to the detection of a particular ligand. Any suitable ligandis contemplated including, but not limited to, IP-10, Mig, I-TAC,MIP-1α, MIP-3α, and MIP-1β. In some embodiments, the ligand is afull-length ligand. In other embodiments, the ligand is a fragment of afull-length ligand. The present invention is not limited to a particularassay. In some embodiments, the reagents comprise reagents forperforming an immunoassay. For example, any suitable immunoassay iscontemplated including, but not limited to, ELISA, radio-immunoassay,automated immunoassay, cytometric bead assay, and immunoprecipitationassay. In some embodiments, the ELISA is a quantitative ELISA assay. Inother embodiments the assay is a Luminex bead assay. In furtherembodiments, the assay is a protein microarray. In some embodiments, thepresent invention further comprises the step of determining the presenceor absence of a concurrent infection in the subject. In someembodiments, the determining of a concurrent infection comprisesdetermining the body temperature of the subject. In other embodiments,the determining of a concurrent infection comprises the detection of abacterial infection in the subject. In still further embodiments, thedetermining of a concurrent infection comprises the detection of a viralinfection in the subject.

In still further embodiments, the present invention provides a kit,comprising reagents for the detection of the amount of a CXCR3 ligand orCCR-5 receptor ligand (e.g., CCL chemokines) in a urine sample from asubject suspected of having acute renal failure, renal tubularinterstitial disease or glomerulonephritis, and instructions for usingthe reagents for detecting the presence of the ligand in the urinesample. The present invention is not limited to the detection of aparticular ligand. Any suitable ligand is contemplated including, butnot limited to, IP-10, Mig, I-TAC, MIP-1α, MIP-3α, and MIP-1β. Thepresent invention is not limited to a particular assay. In someembodiments, the reagents comprise reagents for performing animmunoassay. For example, any suitable immunoassay is contemplatedincluding, but not limited to, ELISA, radio-immunoassay, automatedimmunoassay, cytometric bead assay, and immunoprecipitation assay. Insome embodiments, the ELISA is a quantitative ELISA assay. In otherembodiments the assay is a Luminex bead assay. In further embodiments,the assay is a protein microarray. In some embodiments, the instructionscomprise instructions required by the United States Food and DrugAdministration for use in in vitro diagnostic products. In someembodiments, the kit further comprises second reagents for determiningthe presence or absence of a concurrent infection in the subject andsecond instructions for using the reagent for determining the presenceof absence of the concurrent infection in the subject. In someembodiments, the second instructions comprise instructions fordetermining the body temperature of the subject. In other embodiments,the second reagents comprise reagents for the detection of a bacterialinfection in the subject. In still further embodiments, the secondreagents comprise reagents for the detection of a viral infection in thesubject. In some embodiments, the instructions further compriseinstructions for using the kit for diagnosing tubular interstitialdisease. In other embodiments, the instructions further compriseinstructions for using the kit for predicting the risk of renal tubularinjury.

DESCRIPTION OF THE FIGURES

FIG. 1 shows the design of the Beads FACS method for quantification ofchemokines IP-10, Mig, and I-TAC used in some embodiments of the presentinvention.

FIG. 2 shows urinary levels of CXCR3 binding chemokines IP-10, Mig andI-TAC in recipients of renal allografts, acute tubular injury, BK virusnephritis, borderline rejection, chronic rejection, stable graftfunction and healthy controls.

FIG. 3 shows a comparison of kidney graft recipients with acuterejection, borderline rejection, BK virus nephritis, acute tubularinjury, chronic rejection stable graft function and healthy controlswith urinary chemokine levels greater than 100 pg/mL.

FIG. 4 shows values of IP-10/Mig in differentiation of recipients withacute dysfunction (for example, acute rejection, acute tubular injuryand BK virus nephritis) from recipients with chronic rejection andstable graft function.

FIG. 5 shows the decline of urinary IP-10 and Mig in recipients withacute rejection after anti-rejection therapy.

FIG. 6 shows that urinary IP-10 levels decline several days earlier thanserum creatinine in acute rejection patients receiving anti-rejectiontherapy.

FIG. 7 shows that urine samples from recipients with acute renal graftrejection yield more positive signals for the presence of cytokines,cytokine-related compounds and chemokines, than samples of healthyindividuals, indicating that transplanted kidneys undergoing acuterejection produce cytokines, cytokine-related proteins and chemokinesthat are lacking, or present at much lower levels, in the absence ofacute rejection.

FIG. 8 shows the urine levels (mean, ±standard error) of 23 cytokines,cytokine-related compounds, and chemokines classified into 3 groups (I,II, III) from patient samples (acute renal rejection, acute tubularnecrosis, chronic allograft nephropathy, stable functioning renalgrafts), and from healthy control individuals. Group I factors, whichinclude angiogenin, TIMP-2, TNF sR2 and Trail R3 show a relatively highlevel in the urine samples from all participants. Group II factors,which include IL-1β, IL-2sRα, IL-6, MIP-1α, MIP-1β, MIP-3α, IL-18, andTNF-α show a relatively low level in the urine samples obtained from allparticipants. To the contrary, in comparison the results observed withfactors from Groups I and II, Group III factors, which includeadiponectin, IGFBP-1, IGFBP-2, IGFBP-6, IL-8, leptin, MCP-1, MIP-1δ, TNFsR1, osteoprotogerin (OPG), and uPAR, show low levels of factors in theurine samples of healthy participants, but specific patterns ofelevation for each of the categories of kidney disorder. For example:adiponectin is elevated in all conditions tested; IGFBP-1, IGFBP-2 andIGFBP-6 are elevated in all conditions, but particularly so in acutetubular necrosis and chronic allograft nephropathy; IL-8 is elevated inacute tubular necrosis and stable grafts; leptin is elevated in acutetubular necrosis, but particularly so in acute rejection; MCP-1 iselevated in acute rejection, acute tubular necrosis and to a lesserextent in chronic allograft nephropathy; MIP-1δ is elevated in stablegrafts, but is markedly elevated in acute rejection, acute tubularnecrosis and chronic allograft nephropathy; TNF sR1 is elevated in allkidney disorders tested; and osteoprotogerin (OPG) and uPAR are elevatedin all conditions but particularly so in acute rejection, acute tubularnecrosis, and chronic allograft nephropathy.

FIG. 9 shows that urine levels of IP-10 and Mig are significantlyelevated in samples collected from recipients with acute graft rejection(AR), acute tubular necrosis (ATN) and BK viral nephropathy (BKVN), butnot in samples collected from recipients with borderline rejection,antibody-mediated acute rejection (ABAR), chronic renal allograftrejection (CAN), or in patients with stable graft function (SGF). Urinesamples collected from healthy individuals (HC) contain very low levelsof IP-10 and Mig. FIG. 9 also shows that MIP-1δ and OPG aresignificantly elevated in samples from recipients with AR and ATN, butunlike IP-10 and Mig, MIP-1δ and OPG are also significantly elevated insamples from patients with borderline rejection, ABAR and CAN, but notin patients with BKVN. Similar to the results observed for IP-10 andMig, MIP-1δ and OPG are not elevated in samples collected fromrecipients with stable graft function or from healthy individuals.

FIG. 10 shows that urine levels of IP-10 and Mig differentiate AR, ABAR,ATN and BKVN from CAN and stable graft function with high sensitivityand specificity as depicted in ROC curves. FIG. 10 also shows that urinelevels of MIP-1δ and OPG differentiate AR, ABAR, ATN, BKVN, borderlinerejection and CAN from stable graft function with high sensitivity andspecificity as depicted in ROC curves.

FIG. 11 shows that with 80 pg/ml of IP-10, Mig, MIP-1δ and OPG in urineestablished as a cutoff level (that is, to provide maximal sensitivityand specificity), the majority of renal graft recipients with AR, ABAR,ATN or BKVN have high levels of IP-10 and Mig, while the majority ofrenal graft recipients with CAN or stable graft function have IP-10 andMig levels below the cutoff.

FIG. 12 shows the sensitivity, specificity, positive predictive valueand negative predictive value of IP-10, Mig to differentiate acuteinjury (AR, ABAR, ATN and BKVN) from CAN, stable graft function andhealthy renal function. FIG. 12 also shows that MIP-1δ and OPG are bothhighly sensitive and specific to differentiate acute injury (AR, ABAR,ATN and BKVN), borderline rejection and CAN, from stable graft functionand healthy renal function.

DEFINITIONS

To facilitate an understanding of the present invention, a number ofterms and phrases are defined below:

As used herein, the term “surgical procedure” refers to any procedurethat involves treatment of injury, deformity, or disease by manual orinstrumental means.

As used herein, the term “fluorescently activated cell sorting assay”(FACS) refers to any assay suitable for use in cell sorting techniques(e.g., flow cytometry) that employs detection of fluorescent signals.

As used herein, the terms “immunoglobulin” or “antibody” refer toproteins that bind a specific antigen. Immunoglobulins include, but arenot limited to, polyclonal, monoclonal, chimeric, and humanizedantibodies, Fab fragments, F(ab′)₂ fragments, and includesimmunoglobulins of the following classes: IgG, IgA, IgM, IgD, IbE, andsecreted immunoglobulins (sIg). Immunoglobulins generally comprise twoidentical heavy chains and two light chains. However, the terms“antibody” and “immunoglobulin” also encompass single chain antibodiesand two chain antibodies.

As used herein, the term “antigen binding protein” refers to proteinsthat bind to a specific antigen. “Antigen binding proteins” include, butare not limited to, immunoglobulins, including polyclonal, monoclonal,chimeric, and humanized antibodies; Fab fragments, F(ab′)₂ fragments,and Fab expression libraries; and single chain antibodies.

The term “epitope” as used herein refers to that portion of an antigenthat makes contact with a particular immunoglobulin.

When a protein or fragment of a protein is used to immunize a hostanimal, numerous regions of the protein may induce the production ofantibodies which bind specifically to a given region orthree-dimensional structure on the protein; these regions or structuresare referred to as “antigenic determinants”. An antigenic determinantmay compete with the intact antigen (i.e., the “immunogen” used toelicit the immune response) for binding to an antibody.

The terms “specific binding” or “specifically binding” when used inreference to the interaction of an antibody and a protein or peptidemeans that the interaction is dependent upon the presence of aparticular structure (i.e., the antigenic determinant or epitope) on theprotein; in other words the antibody is recognizing and binding to aspecific protein structure rather than to proteins in general. Forexample, if an antibody is specific for epitope “A,” the presence of aprotein containing epitope A (or free, unlabelled A) in a reactioncontaining labeled “A” and the antibody will reduce the amount oflabeled A bound to the antibody.

As used herein, the terms “non-specific binding” and “backgroundbinding” when used in reference to the interaction of an antibody and aprotein or peptide refer to an interaction that is not dependent on thepresence of a particular structure (i.e., the antibody is binding toproteins in general rather that a particular structure such as anepitope).

As used herein, the term “subject” refers to any animal (e.g., amammal), including, but not limited to, humans, non-human primates,rodents, and the like, which is to be the recipient of a particulardiagnostic test or treatment. Typically, the terms “subject” and“patient” are used interchangeably herein in reference to a humansubject.

As used herein, “cytokine” refers to any of a class of immunoregulatorysubstances (for example, lymphokines) that are secreted by cells of theimmune system. As used herein, “cytokine-related compound” refers to anyof a class of substances that are functionally linked to one or morecytokines, for example, adhesion molecules, selectins, integrins,chemokines, and chemokine receptors.

As used herein. “chemokines” are cytokines characterized, for example,by their ability to induce directed migration of leukocytes, leukocyteactivation and effector function. As used herein, “chemokines” can bedivided into, for example, four branches (C, CC, CXC, and CX3C) basedupon the position of the first two cysteine residues in a four-cysteinemotif in their primary amino acid sequence. As used herein, chemokinesare also classified by their binding characteristics as ligands (L), forexample CL, CCL, CXCL and CX3CL. As used herein, “chemokines” arefurther characterized based on whether they are inflammatory orhomeostatic.

As used herein, in some embodiments “chemokines” are CXCR3 chemokines,including, but not limited to, IP-10, Mig, and I-TAC. In otherembodiments, “chemokines” are CCL class chemokines, which bind to theCCR-5 receptor. Exemplary CCL class chemokines include, but are notlimited to, MIP-1α, MIP-3α, and MIP-1β.

As used herein, the term “acute renal failure” refers to an abrupt andsustained decrease in glomerular filtration, urine output, or both. Forexample, acute renal failure may be an abrupt (1-7 days) and sustained(greater than 24 hours) change from baseline glomerular filtration rate,urine output, or both.

As used herein, “kidney disorder” refers to any pathologic disease orcondition of the kidney including, for example, those diseases andconditions considered in Comprehensive Clinical Nephrology, 2nd Edition,edited by Richard J Johnson and John Feehally, Mosby, 2003, which isincorporated herein by reference in its entirety.

As used herein, “diagnosing disorders of the kidney” refers to, forexample, the detection, identification, monitoring, and screening ofkidney disorders. In some embodiments the diagnosis uses only the assaysof the present invention. In other embodiments, assays of the presentinvention are used for diagnosis of a kidney disorder in combinationwith other indices of kidney function including, for example, patientsigns and symptoms, tests of general kidney function, for example, serumcreatinine and blood urea nitrogen (BUN), or urinalysis, or tests ofspecific disorders of the kidney, for example, kidney biopsy, urine RNAlevels, urine DNA levels, and other urinary markers. In someembodiments, assays of the present invention are used, for example, fordifferential diagnosis between two or more possible diseases orconditions, or for the detection of two or more diagnoses of kidneydisorders in the same patient. In some embodiments, assays of thepresent invention are performed in a health care facility laboratory. Inother embodiments, assays of the present invention are performed in areference clinical laboratory. In further embodiments, assays of thepresent invention are performed at the patient's residence by thepatient, a caregiver, or health care provider.

In some embodiments or the present invention, diagnosing disorders ofthe kidney is based on detecting at least one compound from the listcomprising adiponectin, IGFBP-1, IGFBP-2, IGFBP-6, IL-8, leptin, MCP-1,MIP-1δ, TNF sR1, osteoprotogerin, uPAR, IP-10 and Mig. As used herein,“detecting the presence” and “detecting the amount” of said compoundsrefer to a quantitative or qualitative measures of the compound in theurine of a subject. As used herein, “reagents for detection of at leastone compound” and “reagents for detection of two or more compounds”refer to reagents specific for detection of the cytokines,cytokine-related compounds and chemokines of the present invention. Insome embodiments, the reagent is an antibody. In other embodiments, thereagent is aptamer. In other embodiments, the reagents and kits of thepresent invention further comprise additional reagents and devices forperforming detection assays, including, but not limited to, controls,buffers, and substrates (for example, beads, microspheres, andmicroarrays).

As used herein, the terms “instructions for using said reagents fordetecting the presence of one or more said compounds”, and “instructionsfor using said detecting the presence of one or more said compounds insaid urine sample for diagnosing a kidney disorder” include instructionsfor using the reagents contained in the kit for the diagnosis of akidney disorder in a sample from a subject. In some embodiments, theinstructions further comprise the statement of intended use required bythe U.S. Food and Drug Administration (FDA) in labeling in vitrodiagnostic products. Information required in an application mayinclude: 1) The in vitro diagnostic product name, including the trade orproprietary name, the common or usual name, and the classification nameof the device; 2) The intended use of the product; 3) The establishmentregistration number, if applicable, of the owner or operator submittingthe submission; the class in which the in vitro diagnostic product wasplaced under section 513 of the FD&C Act, if known, its appropriatepanel, or, if the owner or operator determines that the device has notbeen classified under such section, a statement of that determinationand the basis for the determination that the in vitro diagnostic productis not so classified; 4) Proposed labels, labeling and advertisementssufficient to describe the in vitro diagnostic product, its intendeduse, and directions for use, including photographs or engineeringdrawings, where applicable; 5) A statement indicating that the device issimilar to and/or different from other in vitro diagnostic products ofcomparable type in commercial distribution in the U.S., accompanied bydata to support the statement; 6) A summary of the safety andeffectiveness data upon which the substantial equivalence determinationis based; or a statement that the safety and effectiveness informationsupporting the FDA finding of substantial equivalence will be madeavailable to any person within 30 days of a written request; 7) Astatement that the submitter believes, to the best of their knowledge,that all data and information submitted in the premarket notificationare truthful and accurate and that no material fact has been omitted;and 8) Any additional information regarding the in vitro diagnosticproduct requested that is necessary for the FDA to make a substantialequivalency determination. Additional information is available at theInternet web page of the U.S. FDA.

As used herein, the term “determining a treatment course of action” asin “determining a treatment course of action based on said diagnosis ofa kidney disorder” refers to the choice of treatment administered to apatient. For example, if a patient is found to be at increased risk of akidney disorder, therapy may be started, increased, or changed from onetreatment type (e.g., pharmaceutical agent, surgery) to another.Conversely, if a patient is found to be at low risk for a kidneydisorder, therapy may not be administered or levels of therapy may bedecreased. In some embodiments, the treatment course of action is“continued monitoring” in which no treatment is administered but thelevels of cytokines, cytokine-related compounds and chemokines measuredin the patients urine is monitored regularly (e.g., using the diagnosticmethods of the present invention). In other embodiments, the “treatmentcourse of action” as used herein, comprises use of the results of thecytokine, cytokine-related compound and chemokine assays of the presentinvention as indicators of the need for additional tests of a kidneydisorder, for example, an imaging scan, biopsy or ureteroscopic exam.

As used herein, the term “determining the efficacy of said acute renalfailure, renal tubular interstitial disease or glomerulonephritis drugbased on said detecting” refers to determining if a drug is preventingacute renal failure, renal tubular interstitial disease orglomerulonephritis based on, for example, detecting the level ofcytokines, cytokine-related compounds and chemokines in the urine of apatient who manifests signs and symptoms of, or is at risk for acuterenal failure, renal tubular injury or glomerulonephritis.

As used herein, the terms “computer memory” and “computer memory device”refer to any storage media readable by a computer processor. Examples ofcomputer memory include, but are not limited to, RAM, ROM, computerchips, digital video disc (DVDs), compact discs (CDs), hard disk drives(HDD), and magnetic tape.

As used herein, the term “computer readable medium” refers to any deviceor system for storing and providing information (e.g., data andinstructions) to a computer processor. Examples of computer readablemedia include, but are not limited to, DVDs, CDs, hard disk drives,magnetic tape and servers for streaming media over networks.

As used herein, the terms “processor” and “central processing unit” or“CPU” are used interchangeably and refer to a device that is able toread a program from a computer memory (e.g., ROM or other computermemory) and perform a set of steps according to the program.

As used herein, the term “non-human animals” refers to all non-humananimals including, but are not limited to, vertebrates such as rodents,non-human primates, ovines, bovines, ruminants, lagomorphs, porcines,caprines, equines, canines, felines, aves, etc.

“Amino acid sequence” and terms such as “polypeptide” or “protein” arenot meant to limit the amino acid sequence to the complete, native aminoacid sequence associated with the recited protein molecule.

The term “native protein” as used herein to indicate that a protein doesnot contain amino acid residues encoded by vector sequences; that is,the native protein contains only those amino acids found in the proteinas it occurs in nature. A native protein may be produced by recombinantmeans or may be isolated from a naturally occurring source.

As used herein the term “portion” when in reference to a protein (as in“a portion of a given protein”) refers to fragments of that protein. Thefragments may range in size from four amino acid residues to the entireamino acid sequence (that is, the “full size” sequence) minus one aminoacid.

The term “Western blot” refers to the analysis of protein(s) (orpolypeptides) immobilized onto a support such as nitrocellulose or amembrane. The proteins are run on acrylamide gels to separate theproteins, followed by transfer of the protein from the gel to a solidsupport, such as nitrocellulose or a nylon membrane. The immobilizedproteins are then exposed to antibodies with reactivity against anantigen of interest. The binding of the antibodies may be detected byvarious methods, including the use of radiolabeled antibodies.

As used herein, the terms “protein microarray” and “protein chip” referto protein-detecting molecules immobilized at high density on asubstrate, and probed for various biochemical activities. (See, forexample: Zhu H and Snyder M, “Protein chip technology”, Current Opinionin Chemical Biology 7: 55-63, 2003; Cutler P, “Protein arrays: Thecurrent state of the art”, Proteomics 3; 3-18, 2003; and MacBeath G,“Protein microarrays and proteomics”, Nature Genetics Supplement 32:526-532, 2002, each of which is incorporated herein by reference in itsentirety).

As used herein, the term “in vitro” refers to an artificial environmentand to processes or reactions that occur within an artificialenvironment. In vitro environments can consist of, but are not limitedto, test tubes and cell culture. The term “in vivo” refers to thenatural environment (e.g., an animal or a cell) and to processes orreaction that occur within a natural environment.

The terms “test compound” and “candidate compound” refer to any chemicalentity, pharmaceutical, drug, and the like that is a candidate for useto treat or prevent a disease, illness, sickness, or disorder of bodilyfunction (for example, renal acute renal failure, renal tubularinterstitial disease, renal cancer or glomerulonephritis). Testcompounds comprise both known and potential therapeutic compounds. Atest compound can be determined to be therapeutic by screening using thescreening methods of the present invention.

As used herein, the term “sample” is used in its broadest sense. In onesense, it is meant to include a specimen or culture obtained from anysource, as well as biological and environmental samples. Biologicalsamples may be obtained from animals (including humans) and encompassfluids, solids, tissues, and gases. Biological samples include urine andblood products, such as plasma, serum and the like. Such examples arenot however to be construed as limiting the sample types applicable tothe present invention.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to methods of diagnosing, predicting andmonitoring kidney disorders. In particular, the present inventionrelates to the diagnosis, prediction and monitoring of kidney disordersby detection of cytokines, cytokine-related compounds and chemokines inurine. The present invention further relates to methods and compositionsfor assessing the efficacy of agents and interventions used to treatkidney disorders.

For example, the present invention provides a novel, non-invasive methodof correlating the presence of certain cytokines, cytokine-relatedcompounds and chemokines s in urine with acute renal failure, renaltubular interstitial disease, renal cancer or glomerulonephritis. Themethods are a significant improvement over invasive biopsy in terms ofdecreased cost and physical trauma to a patient. The methods of thepresent invention provide the further advantage of allowing home testingby patients.

I. Detection of Cytokines, Cytokine-related Compounds and Chemokines inUrine

In some embodiments, the present invention provides methods ofpredicting and diagnosing a kidney disorder by detecting cytokines,cytokine-related compounds and chemokines in urine. The presentinvention is not limited to a particular detection assay. Thedescription below provides non-limiting examples of suitable cytokines,cytokine-related compounds and chemokines and detection methods. Thepresent invention further provides kits for use in detecting cytokines,cytokine-related compounds and chemokines in urine.

A. Urinary Cytokines, Cytokine-Related Compounds and Chemokines

The present invention provides methods of detecting cytokines,cytokine-related compounds and chemokines in urine. The urinarycytokines, cytokine-related compounds and chemokines of the presentinvention are correlated with the presence or absence of kidneydisorders, for example acute renal failure, renal tubular interstitialdisease, renal cancer or glomerulonephritis. In some embodiments, thepresence of the peptides or an increased amount of the peptides isindicative of tubular injury. In other embodiments, increased urinarycytokines, cytokine-related compounds and chemokines are correlated withincreased risk of acute renal failure, renal interstitial disease, renalcancer or glomerulonephritis. In preferred embodiments, the amount ofurinary cytokine, cytokine-related compound and chemokine isquantitated. In some preferred embodiments, a quantitative level ofurinary cytokine, cytokine-related compound and chemokine is determinedthat is indicative of an increased risk of a kidney disorder. In otherembodiments, the level of cytokine, cytokine-related compound andchemokine is correlated with a functioning level of a drug (e.g., thecorrect amount or a functional drug).

In preferred embodiments, the chemokines are CXCR3 chemokines. CXCR3chemokines include, but are not limited to, IP-10, Mig, and I-TAC. Inother embodiments, the chemokines are CCL chemokines. CCL chemokinesbind to the CCR-5 receptor and include, but are not limited to, MIP-1α,MIP-3α, and MIP-1β. In particularly preferred embodiments, thecytokines, cytokine-related compounds and chemokines are selected fromthe list comprising adiponectin, IGFBP-1, IGFBP-2, IGFBP-6, IL-8,leptin, MCP-1, MIP-1δ, TNF sR1, osteoprotogerin, uPAR, IP-10 and Mig.

In some embodiments, two or more (e.g., 3 or more, 4 or more, etc.)cytokines, cytokine-related compounds and chemokines are detected toprovide a risk assessment. The presence of each marker may provide amore definitive answer than the analysis of any single marker alone. Forexample, as described in Example 2 below, detection of both IP-10 andI-TAC provided a 100% correlation to renal failure in the patient grouptested.

In some embodiments, certain threshold levels of a particular marker aredetected. If the threshold level is reached, risk of acute renalfailure, tubular interstitial disease, renal cancer orglomerulonephritis is observed. For example, if 100 pg/ml of therejection marker (e.g., IP-10, I-TAC) in urine is observed, risk isobserved. Or, for example, if 80 pg/ml of the marker for a kidneydysfunction is reached (for example, adiponectin, IGFBP-1, IGFBP-2,IGFBP-6, IL-8, leptin, MCP-1, MIP-1δ, TNF sR1, osteoprotogerin, uPAR,IP-10 and Mig), increase risk for a kidney disorder is observed. Thepresent invention is not limited by the threshold level used in theanalysis. In some embodiments, the threshold level is 20 pg/ml or more,more preferably, 50 pg/ml or more, preferably 80 mg/ml, and mostpreferably 100 pg/ml or more, although both higher and lower thresholdvalues are contemplated, as are intervals between these values.

B. Detection Methods

The present invention provides methods for detecting the presence ofcytokines, cytokine-related compounds and chemokines in a urine sample.In some embodiments, a full-size cytokine, cytokine-related compound orchemokine polypeptide is detected. In other embodiments, a fragment or aportion of a cytokine, cytokine-related compound or chemokinepolypeptide is detected. In preferred embodiments, the present inventionadditionally provides methods of quantifying the amount of a cytokine,cytokine-related compound and chemokine in urine. The present inventionis not limited to a particular detection assay. In some embodimentsdetection is, for example, fluorescent detection, spectrometricdetection, chemiluminescent detection, matrix assisted laserdesorption-time-of flight (MALDI-TOF) detection, high pressure liquidchromatographic detection, charge detection, mass detection, radiofrequency detection, and light diffraction detection. Exemplarydetection assays are described herein.

In some embodiments, cytokines, cytokine-related compounds andchemokines are detected by binding of a capture molecule specific forthe protein (for example, an aptamer, or an antibody in an immunoassay).The present invention is not limited to a particular capture molecule orantibody. Any capture molecule or antibody (e.g., monoclonal orpolyclonal) that detects cytokines, cytokine-related compounds andchemokines may be utilized. Exemplary methods for the generation ofantibodies are described below.

Antibody binding is detected by techniques known in the art. Forexample, in some embodiments, antibody binding is detected using asuitable technique, including but not limited to, radio-immunoassay,ELISA (enzyme-linked immunosorbant assay), “sandwich” immunoassay,immunoradiometric assay, gel diffusion precipitation reaction,immunodiffusion assay, precipitation reaction, agglutination assay(e.g., gel agglutination assay, hemagglutination assay, etc.),complement fixation assay, immunofluorescence assay, protein A assay,and immunoelectrophoresis assay.

In some preferred embodiments, a quantitative ELISA assay is utilized(See e.g., U.S. Pat. Nos. 5,958,715, and 5,484,707, each of which isherein incorporated by reference). In some preferred embodiments, thequantitative ELISA is a competitive ELISA. In a competitive ELISA, thewells of a microtiter plate are first coated with a fusion proteincomprising all or a fragment of the cytokine, cytokine-related compoundor chemokine (e.g., a CXCR3 or CCL ligand). The sample to be tested isadded to the plate along with an antibody that is specific for thecytokine, cytokine-related compound or chemokine. The cytokine,cytokine-related compound or chemokine in the urine sample competes forbinding to the antibody with the immobilized peptide. The plate iswashed and the antibody bound to the immobilized cytokine,cytokine-related compound or chemokine polypeptide is then detectedusing any suitable method (e.g., a secondary antibody comprising a labelor a group reactive with an enzymatic detection system). The amount ofsignal is inversely proportional to the amount of cytokine,cytokine-related compound or chemokine polypeptide present in the urinesample (e.g., a high signal is indicative of low amounts of cytokine,cytokine-related compound or chemokine polypeptide being present in theurine).

In some embodiments, an automated detection assay is utilized. Methodsfor the automation of immunoassays include, but are not limited to,those described in U.S. Pat. Nos. 5,885,530, 4,981,785, 6,159,750, and5,358,691, each of which is herein incorporated by reference. In someembodiments, the analysis and presentation of results is also automated.For example, in some embodiments, software that generates a diagnosisand/or prognosis based on the level of cytokine, cytokine-relatedcompound or chemokine polypeptide in the urine is utilized. In otherembodiments, the immunoassay described in U.S. Pat. Nos. 5,789,261,5,599,677 and 5,672,480, each of which is herein incorporated byreference, is utilized.

In still other embodiments, a protein microarray or protein chip arrayassay is utilized for detection (See e.g., U.S. Pat. No. 6,197,599,herein incorporated by reference). In such an assay, proteins (e.g.,antibodies specific for a cytokine, cytokine-related compound orchemokine polypeptide) are immobilized on a solid support such as achip. A urine sample suspected of containing the cytokine,cytokine-related compound or chemokine polypeptide is passed over thesolid support. Bound cytokine, cytokine-related compound or chemokinepolypeptides are then detected using any suitable method. In someembodiments, detection is via surface plasmon resonance (SPR) (See e.g.,WO 90/05305, herein incorporated by reference). In SPR, a beam of lightfrom a laser source is directed through a prism onto a biosensorconsisting of a transparent substrate, usually glass, which has oneexternal surface covered with a thin film of a noble metal, which inturn is covered with an organic film that interacts strongly with ananalyte, such as a biological, biochemical or chemical substance. Theorganic film contains antibodies (e.g., specific for a cytokine,cytokine-related compound or chemokine polypeptide of the presentinvention), which can bind with an analyte (e.g., chemokine) in a sampleto cause an increased thickness, which shifts the SPR angle. By eithermonitoring the position of the SPR angle, or the reflectivity at a fixedangle near the SPR angle, the presence or absence of an analyte in thesample can be detected.

In other embodiments, The PROTEINCHIP (Ciphergen Biosystems, Fremont,Calif.) is utilized for detection. The PROTEINCHIP system uses SELDI(Surface-Enhanced Laser Desorption/Ionization) technology to perform theseparation, detection and analysis of proteins at the femtomole leveldirectly from biological samples (See e.g., U.S. Pat. No. 6,294,790 andU.S. Patent Application US20010014461A1, each of which is hereinincorporated by reference. In the PROTEINCHIP technology, proteins ofinterest (e.g., cytokine, cytokine-related compound or chemokinepolypeptides) are captured on the PROTEINCHIP Array (e.g., via a boundantibody) directly from the original source material. The chip is washedto remove undesired materials and bound proteins are detected usingSELDI.

In some embodiments, a cytometric bead array assay is used (Quantum Plexkit, Bangs Laboratories; Cytometric Bead Array kit, BD Biosciences).These systems allow for multiple analyte detection with small volumesamples. In other embodiments, a Luminex bead assay is used.

The present invention is not limited to the detection of cytokines,cytokine-related compounds and chemokines in urine. Any bodily fluidthat contains elevated levels of cytokine, cytokine-related compound andchemokine correlated with a kidney disorder may be utilized, including,but not limited to, blood, serum, lymph, and saliva.

In some particularly preferred embodiments, a combination of severalcytokines, cytokine-related compounds or chemokines are detectedsimultaneously in urine samples. In some embodiments, the presentinvention provides a fluorescently activated cell sorting (FACS) methodfor the simultaneous detection of multiple cytokines, cytokine-relatedcompounds or chemokines. In some embodiments, the method usesfluorescence dye labeled beads that can detect multiple (e.g., at least3) cytokines, cytokine-related compounds or chemokines in one assay. Inone exemplary embodiment (Example 3), the assay was used to detectIP-10, I-TAC and Mig. Detection of these three chemokines was conductedin the same test tube simultaneously as depicted in FIG. 1. As thechemokine concentration increases, the mean fluorescence intensity foreach group of beads increases. This correlation between the chemokineconcentration and the mean fluorescence establishes the basis for thisFACS quantitative method. A standard curve for each chemokine wasconstructed. These results demonstrate a quantitative assay for thesimultaneous detection of multiple cytokines, cytokine-related compoundsand chemokines.

The present invention is further not limited to the direct detection ofcytokine, cytokine-related compound and chemokine polypeptides. Thepresent invention contemplates the detection of correlated polypeptidesor compounds (e.g., cytokine, cytokine-related compound and chemokineDNA, mRNA, metabolites, etc.). In still further embodiments, the presentinvention provides methods of detecting the interaction of cytokines,cytokine-related compounds and chemokines with cytokine,cytokine-related compound and chemokine receptors (e.g., CXCR3 or CCR-5receptors).

C. Detection of Concurrent Infection

In some embodiments, assays for the detection of cytokines,cytokine-related compounds and chemokines are combined with assays forthe detection of concurrent infections (e.g., bacterial or viralinfections) that may generate false-positive results. For example,infection may cause elevated levels of cytokines, cytokine-relatedcompounds and chemokines. In some embodiments, the presence of infectionis monitored along with the presence of cytokines, cytokine-relatedcompounds and chemokines.

In some embodiments, infection is monitored by the presence ofdiagnostic symptoms (e.g., including, but not limited to, elevated bodytemperature, swelling or redness, and pain). In other embodiments,infection is monitored by monitoring the presence of infectiousorganisms such a bacteria, virus, or fungus. In still furtherembodiments, infection is monitored by monitoring the presence ofelevated cytokines, cytokine-related compounds and chemokines that areassociated with infection, but not with a general or specific kidneydisorder. In yet other embodiments, infection is monitored by anelevated white blood cell count in a subject.

D. Kits

In some embodiments, the present invention provides kits for thedetection of cytokines, cytokine-related compounds and chemokines. Insome embodiments, the kits contain antibodies specific for cytokines,cytokine-related compounds and chemokines in addition to detectionreagents, buffers or devices. In some embodiments, the kits containreagents and/or instructions for testing for concurrent infections. Inpreferred embodiments, the kits contain all of the components necessaryto perform a detection assay, including all controls, directions forperforming assays, and any necessary hardware or software for analysisand presentation of results.

In some embodiments, the kits contain an assay in a test strip format.In such embodiments, the detection reagent (e.g., antibody), as well asany control or secondary antibodies, are affixed to a solid support. Insome embodiments, the solid support is a test strip suitable for dippinginto a solution of urine (See e.g., U.S. Pat. Nos. 6,352,862, 6,319,676,6,277,650, 6,258,548, and 6,248,596, each of which is hereinincorporated by reference).

In some embodiments, the kits are marketed as in vitro diagnostics. Themarketing of such kits in the United States requires approval by theFood and Drug Administration (FDA). The FDA classifies in vitrodiagnostic kits as medical devices. The 510(k) regulations specifycategories for which information should be included.

II. Patient Care

The present invention further provides methods of providing test kits topatients in a variety of settings. The test kits of the presentinvention are suitable for use in both clinical and home testingsettings. In preferred embodiments, test kits are approved for sale asin vitro diagnostics as described above.

A. Home Testing

In some embodiments, the present invention provides kits for hometesting. In preferred embodiments, the kits are approved as in vitrodiagnostics for home use under guidelines as described above. Patientsmay use home test kits to monitor acute renal failure, renal tubularinterstitial disease, renal cancer or glomerulonephritis. In someembodiments, test kits for home use are qualitative rather thanquantitative. For example, in some embodiments, the test registers apositive result if urine levels of cytokines, cytokine-related compoundsand chemokines are above a pre-determined level (e.g., aboveapproximately 80 pg/mL) or increase over time. In other embodiments, thetests are quantitative (e.g., utilizing the quantitative methodsdescribed above).

For example, in some embodiments, patients at risk for a kidney disordermonitor urine levels of cytokines, cytokine-related compounds andchemokines. In preferred embodiments, patients conduct serial monitoring(e.g., from once a day to once a month or every several months) toscreen for early signs or renal failure. In preferred embodiments,patients whose urine levels of cytokines, cytokine-related compounds andchemokines are above a pre-determined level (or register a positiveresult in a quantitative assay) are instructed to seek medical advice.

In other embodiments, the test kits are utilized by patients, caregiversor health care providers at the patient's residence to monitor theeffectiveness of a drug. For example, in some embodiments, a patient whois taking a drug following the diagnosis of a kidney disorder monitorslevels of cytokines, cytokine-related compounds and chemokines on aregular basis (e.g., from once a day to once a month or every severalmonths). If a patient's levels of cytokines, cytokine-related compoundsor chemokines are above a pre-determined level (or registers a positiveresult in a quantitative assay), it may be indicative of organ failurecaused by lack of an effective level of a drug. Such patients areadvised to schedule a follow up with a caregiver (e.g., to adjust themedication levels, or switch to a different drug).

B. Clinical-Based Testing

In other embodiments, testing is performed in a clinical (e.g., hospitalor clinic) setting. In such embodiments, testing is generally orderedand interpreted by a physician or other clinician. In some embodiments,testing is carried out by a lab technician (e.g., in an in-house orexternal clinical lab). In preferred embodiments, clinical testingutilizes a quantitative assay for detection of cytokines,cytokine-related compounds and chemokines. In some embodiments, testingis utilized to determine the likelihood of organ failure in a patientwith a kidney disorder. In other embodiments, testing is utilized tomonitor organ function in a subject who has recovered from a kidneydisorder, and is not on medication. In still further embodiments,testing is utilized to monitor the effectiveness of a medication. Insome embodiments, the urinary cytokine, cytokine-related compound orchemokine test is used to complement allograft biopsy and serumcreatinine (Cr), and to monitor response to therapy. In a preferredembodiment, the urinary cytokine, cytokine-related compound andchemokine test is used as a reference parameter in deciding whether andwhen a biopsy should be taken. Combining serum Cr with the urinarycytokines, cytokine-related compounds and chemokines test distinguishesacute dysfunction of the renal allograft, which is medical emergency andneeds urgent treatment, from non-acute elevations of serum creatinine(Cr) and dysfunction. For patients with elevated Cr and, for example,urinary IP-10/Mig, a biopsy should be immediately taken and an accuratediagnosis should be made before the initiation of therapy. In anotherembodiment, in patients who have concurrent elevation of serum Cr andurinary cytokines, cytokine-related compounds or chemokines, if a BKvirus test on urinary cells either by microscopic observation orpolymerase chain reaction is also positive, the likelihood of BK virusnephritis is very high, and a biopsy may be avoided. In a furtherembodiment, if the serum Cr is increased, but there is a normal level ofurinary cytokines, cytokine-related compounds and chemokines, elevationof serum Cr may be due to chronic insidious damage to the renal graft.In this circumstance, a biopsy is delayed or even avoided. In stillfurther embodiments, urine cytokine, cytokine-related compound andchemokine levels are useful in patients whose biopsy reveals borderlinerejection in assessing the need for anti-rejection therapy. In yetfurther embodiments, the urine cytokine, cytokine-related compound andchemokine test distinguishes active low-grade damage in patients havingdormant infiltrating immune cells as found in many biopsies from renalgrafts reveal numerous infiltrating immune cells. These patients oftenhave a normal serum Cr. In additional embodiments, the urinary cytokine,cytokine-related compound and chemokine test is used as an early indexof the response to anti-rejection therapy. In particularly preferredembodiments, the urinary IP-10 test is useful in recipients with acuterejection that is superimposed on chronic injury causing an elevatedbaseline of serum Cr. In these patients serum Cr may not return to thebaseline level, but the urinary IP-10 will decline as acute injuryresolves.

The urinary cytokine, cytokine-related compound and chemokine test ofthe present invention is simple to conduct and rapid, making it suitablefor clinical use. In some embodiments, testing is utilized as a followup to home testing by a patient (e.g., when cytokines, cytokine-relatedcompound and chemokines levels are elevated or the patient has otherclinical signs or symptoms of a kidney disorder). Based on the result ofthe clinical testing, the appropriate intervention is taken (e.g.,including, but not limited to, an increase or decrease in levels of drugtherapy, initiation of drug therapy, termination of therapy, surgery,further testing, or continued monitoring).

C. Home collection/Clinic Testing

In still further embodiments, testing is provided by a clinical lab butin the absence of a physician's order or interpretation. For example, insome embodiments, the patient collects a urine specimen and transportsthe specimen to a clinical lab (e.g., by mail or in person). Theclinical lab then reports the result to the patient. In otherembodiments, the patient provides a sample at a clinical lab, the sampleis analyzed, and the results are returned to the patient. The patientthen decides, based on the level of cytokines, cytokine-relatedcompounds and chemokines in the urine (or the presence or absence of apositive result in a qualitative assay) whether or not to contact aphysician for follow up care.

III. Antibodies

The present invention provides isolated antibodies. In preferredembodiments, the present invention provides monoclonal antibodies thatspecifically bind to an isolated polypeptide comprised of at least fiveamino acid residues of a cytokine, cytokine-related compound orchemokine. These antibodies find use in the diagnostic methods describedherein. In other embodiments, commercially available antibodies areutilized (e.g., available from any suitable source including, but notlimited to, R & D System, Minneapolis, Minn.).

An antibody against a protein of the present invention may be anymonoclonal or polyclonal antibody, as long as it can recognize theprotein. Antibodies can be produced by using a protein of the presentinvention as the antigen according to a conventional antibody orantiserum preparation process.

The present invention contemplates the use of both monoclonal andpolyclonal antibodies. Any suitable method may be used to generate theantibodies used in the methods and compositions of the presentinvention, including but not limited to, those disclosed herein. Forexample, for preparation of a monoclonal antibody, protein, as such, ortogether with a suitable carrier or diluent is administered to an animal(e.g., a mammal) under conditions that permit the production ofantibodies. For enhancing the antibody production capability, completeor incomplete Freund's adjuvant may be administered. Normally, theprotein is administered once every 2 weeks to 6 weeks, in total, about 2times to about 10 times. Animals suitable for use in such methodsinclude, but are not limited to, primates, rabbits, dogs, guinea pigs,mice, rats, sheep, goats, etc.

For preparing monoclonal antibody-producing cells, an individual animalwhose antibody titer has been confirmed (e.g., a mouse) is selected, and2 days to 5 days after the final immunization, its spleen or lymph nodeis harvested and antibody-producing cells contained therein are fusedwith myeloma cells to prepare the desired monoclonal antibody producerhybridoma. Measurement of the antibody titer in antiserum can be carriedout, for example, by reacting the labeled protein, as describedhereinafter and antiserum and then measuring the activity of thelabeling agent bound to the antibody. The cell fusion can be carried outaccording to known methods, for example, the method described by Koehlerand Milstein (Nature 256:495 [1975]). As a fusion promoter, for example,polyethylene glycol (PEG) or Sendai virus (HVJ), preferably PEG is used.

Examples of myeloma cells include NS-1, P3U1, SP2/0, AP-1 and the like.The proportion of the number of antibody producer cells (spleen cells)and the number of myeloma cells to be used is preferably about 1:1 toabout 20:1. PEG (preferably PEG 1000-PEG 6000) is preferably added inconcentration of about 10% to about 80%. Cell fusion can be carried outefficiently by incubating a mixture of both cells at about 20° C. toabout 40° C., preferably about 30° C. to about 37° C. for about 1 minuteto 10 minutes.

Various methods may be used for screening for a hybridoma producing theantibody (e.g., against a CXCR3 or CCL ligand). For example, where asupernatant of the hybridoma is added to a solid phase (e.g.,microplate) to which antibody is adsorbed directly or together with acarrier and then an anti-immunoglobulin antibody (if mouse cells areused in cell fusion, anti-mouse immunoglobulin antibody is used) orProtein A labeled with a radioactive substance or an enzyme is added todetect the monoclonal antibody against the protein bound to the solidphase. Alternately, a supernatant of the hybridoma is added to a solidphase to which an anti-immunoglobulin antibody or Protein A is adsorbedand then the protein labeled with a radioactive substance or an enzymeis added to detect the monoclonal antibody against the protein bound tothe solid phase.

Selection of the monoclonal antibody can be carried out according to anyknown method or its modification. Normally, a medium for animal cells towhich HAT (hypoxanthine, aminopterin, thymidine) are added is employed.Any selection and growth medium can be employed as long as the hybridomacan grow. For example, RPMI 1640 medium containing 1% to 20%, preferably10% to 20% fetal bovine serum, GIT medium containing 1% to 10% fetalbovine serum, a serum free medium for cultivation of a hybridoma(SFM-101, Nissui Seiyaku) and the like can be used. Normally, thecultivation is carried out at 20° C. to 40° C., preferably 37° C. forabout 5 days to 3 weeks, preferably 1 week to 2 weeks under about 5% CO₂gas. The antibody titer of the supernatant of a hybridoma culture can bemeasured according to the same manner as described above with respect tothe antibody titer of the anti-protein in the antiserum.

Separation and purification of a monoclonal antibody (e.g., against aCXCR3 or CCL ligand) can be carried out according to the same manner asthose of conventional polyclonal antibodies such as separation andpurification of immunoglobulins, for example, salting-out, alcoholicprecipitation, isoelectric point precipitation, electrophoresis,adsorption and desorption with ion exchangers (e.g., DEAE),ultracentrifugation, gel filtration, or a specific purification methodwherein only an antibody is collected with an active adsorbent such asan antigen-binding solid phase, Protein A or Protein G and dissociatingthe binding to obtain the antibody.

Polyclonal antibodies may be prepared by any known method ormodifications of these methods including obtaining antibodies frompatients. For example, a complex of an immunogen (an antigen against theprotein) and a carrier protein is prepared, and an animal is immunizedby the complex according to the same manner as that described withrespect to the above monoclonal antibody preparation. A materialcontaining the antibody against is recovered from the immunized animaland the antibody is separated and purified.

As to the complex of the immunogen and the carrier protein to be usedfor immunization of an animal, any carrier protein and any mixingproportion of the carrier and a hapten can be employed as long as anantibody against the hapten, which is crosslinked on the carrier andused for immunization, is produced efficiently. For example, bovineserum albumin, bovine cycloglobulin, keyhole limpet hemocyanin, etc. maybe coupled to an hapten in a weight ratio of about 0.1 part to about 20parts, preferably, about 1 part to about 5 parts per 1 part of thehapten.

In addition, various condensing agents can be used for coupling of ahapten and a carrier. For example, glutaraldehyde, carbodiimide,maleimide-activated ester, activated ester reagents containing thiolgroup or dithiopyridyl group, and the like find use with the presentinvention. The condensation product as such or together with a suitablecarrier or diluent is administered to a site of an animal that permitsthe antibody production. For enhancing the antibody productioncapability, complete or incomplete Freund's adjuvant may beadministered. Normally, the protein is administered once every 2 weeksto 6 weeks, in total, about 3 times to about 10 times.

The polyclonal antibody is recovered from blood, ascites and the like,of an animal immunized by the above method. The antibody titer in theantiserum can be measured according to the same manner as that describedabove with respect to the supernatant of the hybridoma culture.Separation and purification of the antibody can be carried out accordingto the same separation and purification method of immunoglobulin as thatdescribed with respect to the above monoclonal antibody.

The protein used herein as the immunogen is not limited to anyparticular type of immunogen. For example, a cytokine, cytokine-relatedcompound or chemokine polypeptide (further including a gene having anucleotide sequence partly altered) can be used as the immunogen.Further, fragments of the protein may be used. Fragments may be obtainedby any methods including, but not limited to expressing a fragment ofthe gene, enzymatic processing of the protein, chemical synthesis, andthe like.

IV. Drug Screening

In some embodiments, the present invention provides drug-screeningassays (e.g., to screen for drugs effective in treating disorders of thekidney). The screening methods of the present invention utilize thedetection of cytokines, cytokine-related compounds and chemokines. Forexample, in some embodiments, the present invention provides methods ofscreening for compounds that alter (e.g., increase or decrease) theexpression of cytokines, cytokine-related compounds and chemokines. Insome embodiments, the levels of cytokines, cytokine-related compoundsand chemokines are detected (e.g., using a method described herein) in asubject that has undergone administration of a candidate compound. Theincreased levels of cytokines, cytokine-related compounds and chemokinesare indicative of a candidate compound that is not preventing renalfailure. Conversely, preferred candidate compounds are those thatnormalize cytokine, cytokine-related compound and chemokine levels.

In some embodiments, drug screening assays are performed in animals. Anysuitable animal may be used including, but not limited to, baboons,rhesus or other monkeys, mice, or rats. Animal models of kidneydisorders are generated (e.g., by the administration of compounds thattrigger renal failure), and the effects of candidate drugs on theanimals are measured. In preferred embodiments, kidney disorders in theanimals are measured by detecting levels of cytokines, cytokine-relatedcompounds and chemokines in the urine of the animals. The level ofcytokines, cytokine-related compounds and chemokines may be detectedusing any suitable method, including, but not limited to, thosedisclosed herein.

EXPERIMENTAL

The following examples are provided in order to demonstrate and furtherillustrate certain preferred embodiments and aspects of the presentinvention, and are not to be construed as limiting the scope thereof.

Example 1 Correlation of Urine IP-10 with Graft Rejection

This example describes the correlation of urine levels of IP-10 withkidney graft rejection in human subjects. Forty-five human subjects thathad undergone kidney transplant were investigated. Urine IP-10 levelswere measured serially after organ transplantation. IP-10 levels weremeasured using a quantitative calorimetric sandwich ELISA assay (R & DSystems, Minneapolis, Minn.). Subjects were divided into two groups,rejecters and non-rejecters, based on kidney biopsies. Biopsies wereclassified using Banff criteria. All subjects were receivinganti-rejection therapy at the time of the study. Urine from ten normal(non-transplant) subjects was also tested.

In a majority of the non-rejecters, IP-10 levels remained at a constant,low level or decreased over time. In the rejecters, IP-10 levelsremained at a constant, high level or increased over time. A urine levelof IP-10 of greater than approximately 100 pg/mL was associated withorgan rejection. There was no detectable IP-10 in any of the normalcontrol samples. This Example demonstrates that IP-10 levels arecorrelated with kidney transplant rejection.

Experiments conducted during the development of the present inventionalso demonstrated a correlation between CCL chemokines and rejection.For example, correlations were observed for the CCL chemokines MIP-1α,MIP-3α, and MIP-1β.

Example 2 Correlation of Urinary Chemokines with Graft Rejection andTreatment

This example describes the correlation of urinary chemokines levels withgraft rejection and treatment. Urinary samples were collected fromhealthy individuals, kidney transplant recipients with stable graftfunction, and recipients with acute rejection. All patients with acuterejection were hospitalized and received anti-rejection therapy. Urinarysamples were centrifuged, and supernatant was aliquoted and stored at−80° C. These samples, after thawing, were evaluated by ELISA for theexpression of MCP-1, IP-10, and I-TAC.

Elevated Expression of Chemokines in Urinary Samples from Patients withAcute Rejection

As shown in Table 1, chemokines IP-10 and I-TAC were significantlyincreased in the urinary samples of patients with acute graft rejectionand acute tubular injury, compared to healthy controls and kidneytransplant patients with other pathologic changes. As presented in Table2, if 100 pg/ml was used as the cut-off level and IP-10 and I-TAC wereconsidered simultaneously, 80% of samples from patients with rejectionand acute tubular injury were above this level, but less than 5% of thepatients in the remaining groups were above this level. This resultindicates that detection of IP-10 and I-TAC in the urinary samplesreflects the acute rejection/acute tubular injury in the kidney grafts.

MCP-1 was also examined in the urinary samples. In the present series ofsamples (Table 1), urinary MCP-1 was increased in patients with acuterejection or acute tubular injury. However, the difference of MCP-1levels between acute rejection/acute tubular injury and the remaininggroups of patients was not significant.

TABLE 1 Urinary Chemokine Levels in Patients with Kidney TransplantAcute Others (non- Acute Suspicious Rejection Healthy acute/ChronicChronic Tubular Acute (n = 10) Controls Rejection; Rejection InjuryRejection Day 1/ (n = 10) n = 16) (n = 7) (n = 3) (n = 7) Day 2* IP-10(pg/ml) 1 12 31 362 27.8 376/579 I-TAC (pg/ml) 1 21 13 75 44 94.2/168 MCP-1 (pg/ml) 269 641 1908 3226 528 2060/2473 *Day 1/Day 2 indicates thebiopsy day/the day after the biopsy day.

TABLE 2 Patients with Urinary IP-10 and I-TAC Levels above 100 pg/mlOthers (non- Acute Suspicious Acute Healthy acute/Chronic ChronicTubular Acute Rejection Controls Rejection; Rejection Injury Rejection(n = 10) (n = 10) n = 16) (n = 7) (n = 3) (n = 7) Day1/Day* IP-10 0 0 03 1 7/6 I-TAC 0 0 0 1 0 4/6 IP-10 or 0 0 0 3 1 8/8 I-TAC *Day 1/Day 2indicates the biopsy day/the day after the biopsy day.Return of Urinary Chemokine Levels to Baseline after Resolution of AcuteRejection

Urinary samples were collected daily from patients with biopsy-provenacute graft rejection until the rejection resolved. IP-10 and I-TAC weredetermined in these samples with ELISA. IP-10 and I-TAC were elevated atthe time of diagnosis, but the levels decreased after anti-rejectiontherapy was started, and finally returned to the baseline. These resultsindicate that chemokine levels are useful parameters for monitoring thetherapeutic response to anti-rejection therapies. In contrast, MCP-1levels did not return to baseline in at least 50% of the patients withacute rejection following successful treatment. This example indicatesthat IP-10 and I-TAC levels correlate with acute rejection processes inthe kidney graft.

Example 3 Flow Cytometry Based Technique for Quantification ofChemokines

This example describes a FACS method for the simultaneous detection ofmultiple chemokines. The fluorescence activated cell sorting (FACS)method uses fluorescence dye labeled beads that can detect 3 chemokinesin one assay. In this example, IP-10, I-TAC and Mig were detected.Detection of the three chemokines was conducted in the same test tubesimultaneously as depicted in FIG. 1. As the chemokine concentrationincreases, the mean fluorescence intensity for each group of beadsincreases. This correlation between the chemokine concentration and meanfluorescence establishes the basis for the FACS quantitative method. Astandard curve for each chemokine was constructed. This exampledemonstrates that IP-10, Mig and I-TAC can be simultaneously detected ina urinary sample.

Example 4 Urinary Chemokine Assay using the Luminex Microsphere PlatformSubjects

Ninety-nine renal allograft recipients were recruited and donated 350urinary samples. Among the patients, 28 were diagnosed with acuterejection, 9 with borderline rejection, 6 with BK virus nephritis, 10with acute tubular injury, 20 with chronic rejection, and 26 with stablegraft function without graft injury. Urinary samples were also collectedfrom 16 healthy non-transplanted individuals. Renal transplantrecipients with symptoms and elevated serum creatinine (Cr) underwentrenal transplant biopsy which was used as the diagnostic standard. Acuteand chronic rejection events were scored according to Banff criteria bya renal transplant pathologist. BK virus infection was diagnosed usinglight microscopy identification of pleomorphic, enlarged tubularepithelial cell nuclei containing characteristic “smudgy” inclusions.The suspicion of BK virus infection was confirmed in all cases byimmunohistochemistry using a polyclonal polyoma virus-reactive antibody.Urine samples (50 ml) were collected prior to biopsy by clean catch fromthe renal transplant recipients who had an elevated Cr of 20% or moreabove baseline and who were to undergo renal transplant biopsy as adiagnostic procedure. Patients with biopsy proven acute rejection werehospitalized, treated with anti-rejection therapy, and donated dailyurinary samples until the rejection resolved. The collected samples werecentrifuged at 1500 rpm for 10 min. The supernatant of each sample wasaliquoted and stored at −80° C. until use. At the time of experiments,samples were thawed and evaluated for the levels of IP-10, Mig andI-TAC.

Quantification of Urinary IP-10, Mig and I-TAC

Luminex (Austin, Tex.) Multi-Analyte Profiling (xMAP) Technology andRenovar (Madison, Wis.) human CXCR3 binding chemokines triplex assaykits were used for quantification of urinary IP-10, Mig and I-TAC.Luminex xMAP is based on polystyrene particles (microspheres) that areinternally labeled with 2 different fluorophores. When excited by a635-nm laser, the fluorophores emit light at different wavelengths, 658and 712 nm. By varying the 658-nm/712-nm emission ratios, the beads areindividually classified by the unique Luminex 100 IS analyzer. A thirdfluorophore coupled to a reporter molecule allows for quantification ofthe interaction that has occurred on the microsphere surface. Thecapture antibodies directed respectively to IP-10, Mig and I-TAC wereseparately pre-conjugated to their corresponding particles following theLuminex coupling protocol. The quantification of CXCR3 bindingchemokines was conducted in 96-well flat-bottom plates. Twenty-five μlof mixed IP-10, Mig and I-TAC standards or urinary samples were added towells containing 25 μl of assay buffer and 25 μl of pre-coatedparticles, and incubated on a 3-D rotator (Labline Instrument Inc.,Melrose Park, Ill.) at 60 rounds/min at room temperature (RT) for 60min. Mixed biotin labeled detection antibodies directed at IP-10 (BDPharMingen, San Jose, Calif.), Mig and I-TAC (R&D Systems, Minneapolis,Minn.) were then added and incubated on the rotator at 60 rounds/min atRT for 60 min before the addition of streptavidin-PE (BD PharMingen).After an additional 30 min incubation on the rotator at 60 rounds/min atRT, data acquisition and analysis were performed on a Luminex 100 ISanalyzer.

Statistical Analysis

The levels of urinary IP-10, Mig and I-TAC were expressed as meanvalue±standard error (SE). The statistical significance of the findingswas assessed by ANOVA using computer software Prism 4 from GraphPadSoftware (San Diego, Calif.), and a p value less than 0.05 wasconsidered significant. The urinary chemokine threshold that gave themaximal sensitivity and specificity for the diagnosis of acutedysfunction of renal allograft was 100 pg/ml. Sensitivity, specificity,positive predictive value (PPV) and negative predictive value (NPV) ofthe urinary chemokine test was calculated as follows: sensitivity=numberof true positive specimens (TP)/[TP+number of false negative specimens(FN)]; specificity=number of true negative specimens (TN)/[TN+number offalse positive specimens (FP)]; PPV=TP/(TP+FP), and NPV=TN/(TN+FN).

Levels of Urinary Chemokines in Patients and Control Individuals

Urinary IP-10, Mig and I-TAC were simultaneously quantified in eachurine sample by the Luminex xMAP method. FIG. 2 shows that urinarylevels of CXCR3-binding chemokines IP-10, Mig and I-TAC weresignificantly elevated (P<0.01) in samples collected from recipientswith acute rejection (AR), BK virus nephritis (BK VN), and acute tubularinjury (ATI), but not in samples collected from recipients withborderline rejection (BR), chronic rejection (CR), and stable graftfunction (SGF). Furthermore, urinary samples collected from healthycontrol (HC) individuals contained very low levels of the measuredchemokines (FIG. 2).

Urinary Chemokines as a Sensitive and Specific Indicator of Acute RenalAllograft Dysfunction

After renal transplantation, graft dysfunction may occur due totreatable etiologies such as acute rejection, acute tubular injury, orBK virus nephritis. Other types of graft injury may occur moreinsidiously and usually do not require acute intervention, such aschronic rejection and recurrence of the original disease. Acutedysfunction refers herein to acute rejection, acute tubular injury andBK virus nephritis. Levels of urinary IP-10, Mig and I-TAC variedgreatly among the study subjects, ranging from 0 pg/ml to 2000 pg/ml.Using 100 pg/ml as the cutoff level for the urinary chemokines that gavethe maximal sensitivity and specificity, as presented in FIG. 3, most ofthe renal graft recipients with acute rejection, BK virus nephritis andacute tubular injury had higher levels of urinary IP-10 and Mig, whilemost of the recipients with chronic rejection and stable graft functionhad lower levels of urinary IP-10 and Mig (FIG. 3). For recipients withborderline rejection, 4 out of 9 cases showed higher levels. None of thehealthy controls had urinary IP-10, Mig or I-TAC higher than 100 pg/ml.The elevation of urinary IP-10 and Mig was more prevalent than I-TAC inrecipients with acute dysfunction caused by acute rejection, acutetubular injury and BK virus nephritis. As shown in FIG. 2 and FIG. 3,elevation of urinary IP-10 and Mig indicated acute renal injury by oneof the three etiologies. To evaluate the value of urinary IP-10 and/orMig to differentiate the acute dysfunction from chronic rejection andstable graft function, we calculated the sensitivity, specificity,positive predictive value and negative predictive value as presented inFIG. 4. Both IP-10 and Mig are highly sensitive and specific.

Urinary chemokine levels were compared to the renal function indicator,serum Cr. When urinary IP-10 was used in the analysis, most of therecipients with acute dysfunction had increased urinary IP-10 and serumCr. Recipients with chronic rejection had increased Cr, but not IP-10.Recipients with stable graft function had low IP-10 and Cr levels.

Decline of Urinary Chemokines after Anti-Rejection Therapy

Among the 28 patients with acute rejection, daily urinary samples werecollected from 24 during hospitalization for anti-rejection therapy. Day1 is the time that acute rejection was diagnosed and anti-rejectiontherapy was initiated. As presented in FIG. 5, urinary IP-10 and Migdeclined with the initiation of anti-rejection therapy on day 1, andmost of the recipients reached a level below 100 pg/ml in their lastcollected urinary sample.

Serum Cr is an important parameter used to judge the effectiveness ofanti-rejection therapy. Therefore, we compared the levels of serum Crand urinary chemokines in the recipients who received anti-rejectiontherapy. As shown in FIG. 6, urinary IP-10 (solid triangle) declinedseveral days earlier than serum creatinine (solid square) in acuterejection patients receiving anti-rejection therapy. Urinary and serumsamples were collected once daily from each of the patients, and urinaryIP-10 and serum Cr were separately determined. UCR2, UCR5, UCR23, andUCR 12 are representative patients that had elevated serum Cr initiallywhich declined with anti-rejection therapy. UCR20 and UCR22 arerepresentative patients that had elevated serum Cr initially, but Cr didnot decline with anti-rejection therapy during the hospitalizationperiod. Thus, as shown in FIG. 6, the serum Cr in the recipients whowere hospitalized for 3-14 days could be classified into two patterns:started high and remained high for several days before declining(represented by UCR2, UCR5, UCR23, and UCR 12), or started at the lowerrange of the abnormal level and maintained that level (represented byUCR20 and UCR22) during the hospitalization. In contrast to serum Crkinetics, urinary IP-10 declined in all patients, with this decrementstarting 2-5 days earlier than the serum Cr (FIG. 6).

Example 5 Diagnosis of Kidney Disorders Using Levels of Urine Cytokine,Cytokine-Relate Compounds and Chemokines Subjects

Renal transplant patients were recruited from the Transplant Clinic ofthe University of Wisconsin Hospital and Clinics. The research protocolwas approved by the University of Wisconsin Institutional Review Board,and all patients provided informed consent. On the day of biopsy, urinesamples (50 ml) were collected prior to biopsy by clean catch frompatients who had an elevated serum creatinine (Cr) of 20% or more abovebaseline, and who were to undergo renal transplant biopsy as adiagnostic procedure. One hundred and thirteen renal allograftrecipients were recruited. Among the patients, 37 were diagnosed asacute rejection (AR), 10 as borderline rejection, 4 as antibody-mediatedacute rejection (ABAR), 4 as BK virus nephropathy (BKVN), 9 as acutetubular necrosis (ATN), 20 as chronic allograft nephropathy (CAN), and29 as stable graft function. All patients except those with stable graftfunction had a biopsy and pathologic diagnosis. Urine samples were alsocollected from 19 healthy non-transplanted individuals.

The urine samples were centrifuged at 1500 rpm for 10 min. Thesupernatant of each sample was aliquoted and stored at −80° C. untiluse. At the time of experiments, the samples were thawed and evaluated.Patients with signs and symptoms of a kidney disorder and/or elevatedserum Cr underwent renal transplant biopsy. Acute and chronic rejectionwas scored according to Banff criteria by an experienced renaltransplant pathologist. BKVN was diagnosed by light-microscope viaidentification of pleomorphic, enlarged tubular epithelial cell nucleicontaining characteristic “smudgy” inclusions. The diagnosis of BKVN wasconfirmed by immunohistochemistry using a polyclonal polyomavirus-reactive antibody.

Screening Urinary Samples with a Cytokine/Cytokine-RelatedCompound/Chemokine Array

Three urinary samples from renal transplant recipients and 3 samplesfrom healthy controls were used for the initial screening assay usingRayBio® Human Cytokine Antibody Array C Series 1000 kit (RayBiotech,Norcross, Ga.). This antibody array detects and measures 120 cytokines,cytokine-related compounds and chemokines. The manufacturer's suggestedexperimental procedures were followed. Briefly, after blocking andwashing the array membrane, 2 ml of urine sample were mixed with 2 ml ofreaction buffer, and added onto the array membrane and incubated at 4°C. overnight. The reaction buffer is described in U.S. patentapplication Ser. No. 10/968,597, “Reagents for Urine-Based ImmunologicalAssay”, filed Oct. 19, 2004, which is incorporated herein by referencein its entirety. The array membrane was then washed 3 times, followed byincubation with the biotin labeled detection antibody mixture at roomtemperature (RT) for 2 hours. After washing, 2 ml of 1000 fold dilutedHRP-conjugated streptavidin were applied to the array membrane andincubated at RT for 2 hours. Detection reagent was added to the arraymembrane after washing away the unbound HRP-conjugated streptavidin.Signal detection was done by exposing an X-ray film to the arraymembrane.

Screening Urinary Samples with a Cytokine/Cytokine-RelatedCompound/Chemokine Array

In experiments performed with the RayBio® Human Cytokine Antibody ArrayC Series 1000 kit, 23 cytokines, cytokine-related compounds orchemokines were found to have higher signals in the urinary samplesderived from the renal graft rejection patients than in the samples ofhealthy controls (FIG. 7). Acute rejection (AR) is the most common acutecomplication after kidney transplantation, exhibiting severeinflammation mediated by immune reaction. As shown in FIG. 7, urinesamples derived from kidney recipients with AR yield much more positivesignals compared to the samples of healthy individuals, indicating thatthe transplanted kidneys with AR produce cytokines, cytokine-relatedcompounds and chemokines that are not made or made much less by thehealthy kidney. By comparing the screening results, 23 cytokines,cytokine-related compounds and chemokines that appeared with strongersignals in the AR urine samples were selected for further evaluation(FIG. 8). Besides these 23 cytokines, cytokine-related compounds andchemokines, Mig also presented a stronger signal in the AR samples.Because CXCR3 binding chemokines in urine samples of renal transplantrecipients were previously validated, Mig and IP-10 were notspecifically incorporated into the Example 6 screening assay, but wereincluded in the multiplex (that is, quadraplex) assays (FIGS. 9, 10 and11).

Example 6 Further Screening the Cytokine and Chemokine Markers in Urineby a Luminex Microarray

In experiments performed with the RayBio® Human Cytokine Antibody ArrayC Series 1000 kit, 23 cytokines, cytokine-related compounds andchemokines were found with higher signal intensity in urine samplesderived from kidney rejection patients than in the samples of healthycontrols (FIG. 7). These 23 cytokines/chemokines were further screenedusing a microarray developed on the Luminex (Austin, Tex.) Multi-AnalyteProfiling (xMAP) platform. The Luminex xMAP platform is based onpolystyrene particles (microspheres) that are internally labeled with 2different fluorophores. When excited by a 635-nm laser, the fluorophoresemit light at two wavelengths, 658 and 712 nm. By varying the658-nm/712-nm emission ratios, the beads can be individually classifiedby the unique Luminex 100 IS analyzer. A third fluorophore coupled to areporter molecule allows for quantification of the interaction that hasoccurred on the microsphere surface.

Twenty-three capture antibodies directed separately at the 23 cytokines,cytokine-related compounds and chemokines (FIG. 8) were purchased fromR&D Systems (R&D Systems, Minneapolis, Minn.), and were separatelyconjugated to 23 distinct Luminex beads following the manufacturer'sbead conjugation protocol. After confirming that the conjugation wassuccessful, the 23 types of conjugated beads were mixed at a 1:1 ratiofor the microarray assay. The experiment was conducted on a 96-wellplate. Twenty-five μl of urinary samples were added to wells containing25 μl of assay buffer and 25 μl of the pre-coated beads, and incubatedon a 3-D rotator (Labline Instrument Inc., Melrose Park, Ill.) at 60rounds/min at RT for 60 min. The assay buffer is described in U.S.patent application Ser. No. 10/968,597, “Reagents for Urine-BasedImmunological Assay”, filed Oct. 19, 2004, which is incorporated hereinby reference in its entirety. Mixed biotin labeled detection antibodiesdirected at the 23 cytokines/chemokines (R&D Systems, Minneapolis,Minn.) were then added and incubated on the rotator at 60 rounds/min atRT for 60 min. After vacuuming and washing, streptavidin-PE (BDPharMingen) (San Diego, Calif.) was added, and incubated on the rotatorat 60 rounds/min at RT for 30 min. Data acquisition and analysis wereperformed on a Luminex 100 IS analyzer.

Quantification of Urinary IP-10, Mig, MIP-1δ and OPG

The Luminex xMAP Technology and Renovar human cytokine, cytokine-relatedcompound and chemokines quadruplex assay kit (Madison, Wis.) were usedfor simultaneous quantification of urinary IP-10, Mig, MIP-1δ and OPG.The capture antibodies directed respectively at IP-10, Mig, MIP-1δ andOPG were separately pre-conjugated to their corresponding particlesfollowing the Luminex coupling protocol. Quantification of CXCR3 bindingchemokines was conducted in 96-well flat-bottom plates. Twenty-five μlof mixed IP-10, Mig, MIP-1δ and OPG standards or urinary samples wereadded to wells containing 25 μl of assay buffer and 25 μl of pre-coatedparticles, and incubated on a 3-D rotator (Labline Instrument Inc.,Melrose Park, Ill.) at 60 rounds/min at RT for 60 min. Mixed biotinlabeled detection antibodies directed at IP-10 (BD PharMingen, San Jose,Calif.), Mig, MIP-1δ and OPG (R&D Systems, Minneapolis, Minn.) were thenadded and incubated on the rotator at 60 rounds/min at RT for 60 minbefore the addition of streptavidin-PE (BD PharMingen). After anadditional 30 min incubation on the rotator at 60 rounds/min at RT, dataacquisition and analysis were performed on a Luminex 100 IS analyzer.

Statistical Analysis

The levels of urinary IP-10, Mig, MIP-1δ and OPG were expressed as meanvalue±standard error (SE). The statistical significance of thecomparisons was assessed by ANOVA using computer software Prism 4 fromGraphPad Software (San Diego, Calif.), and a p value less than 0.05 wasconsidered significant. The urinary cytokine, cytokine-related compoundor chemokine threshold that gave maximal sensitivity and specificity forthe diagnosis of acute/chronic dysfunction of renal allograft wasdetermined by using a receiver operation characteristics (ROC) curve.Sensitivity, specificity, positive predictive value (PPV) and negativepredictive value (NPV) of the urine tests were calculated as follows:sensitivity=number of true positive specimens (TP)/[TP+number of falsenegative specimens (FN)]; specificity=number of true negative specimens(TN)/[TN+number of false positive specimens (FP)]; PPV=TP/(TP+FP), andNPV=TN/(TN+FN).

Further Screening the Cytokine and Chemokine Markers in Urine by aLuminex Microarray

The Luminex xMAP array was chosen to further evaluate the 23 cytokines,cytokine-related compounds and chemokines derived from the RayBio arrayassay. As shown in FIG. 8, multiple samples were obtained forclassification of the 23 cytokines, cytokine-related compounds andchemokines into 3 groups. Group I includes angiogenin, TIMP-2, TNF sR2and Trail R3. These factors are expressed at relatively high levels inurine samples from patients with AR, ATN, CAN and stable graft function,and from the healthy individuals. Group II includes IL-1, IL-2sRα, IL-6,MIP-1α, MIP-1β, MIP-3α, IL-18, and TNF-α. These factors are expressedrelatively low levels in urine samples from renal transplant recipients,and healthy individuals. Group III includes adiponectin, IGFBP-1,IGFBP-2, IGFBP-6, IL-8, leptin, MCP-1, MIP-1δ, TNF sR1, osteoprotogerin(OPG), and uPAR. These markers are expressed at higher levels in urinesamples from renal transplant recipients with AR, ATN, and CAN than inurine samples derived from kidney recipients with stable graft function,and from healthy individuals.

Levels of Urinary IP-10, Mig, MIP-1δ and OPG in Patients and ControlIndividuals

After two rounds of screening assays, adiponectin, IGFBP-1, IGFBP-2,IGFBP-6, leptin, MIP-1δ, OPG, and uPAR exhibited the highest correlationwith the diagnosis of kidney disorders. For a more detailed evaluation,a quadruplex Luminex xMAP assay including IP-10, Mig, MIP-1δ and OPG wasdeveloped. This quadruplex Luminex xMAP assay simultaneously measuresthe concentration of IP-10, Mig, MIP-1δ and OPG in urine. As shown inFIG. 9, urine IP-10 and Mig are significantly elevated in samplescollected from recipients with AR, BKVN, and ATN, but not in samplescollected from recipients with borderline rejection, ABAR, chronicrejection, and stable graft function. Urine samples collected fromhealthy individuals contained very low levels of IP-10 and Mig. MIP-1δand OPG were significantly elevated in samples collected from kidneyrecipients with AR, borderline rejection, ABAR, ATN, and CAN, but not insamples collected from recipients with stable graft function and healthyindividuals.

Urine IP-10, Mig, MIP-1δ and OPG as Indices of Renal AllograftDysfunction

Acute graft dysfunction after transplant may arise from diverseetiologies, for example, AR, ATN, or BK viral nephropathy. Urgent andspecific intervention tailored to each diagnosis is often required.Other types of graft injury, for example borderline rejection and CAN atearly stage, may be insidious. The latter cases may not require urgenttreatment, but early intervention (for instance, discontinue renal-toxicimmunosuppressive agents and treat borderline rejection) may protect thegraft from further damage. Early detection and specific diagnosis ofthese kidney disorders is therefore essential to the protection of therenal grafts and their recipients.

The present data shows that the quadraplex assays of the presentinvention have the capacity to detect, identify and differentiate acuteand chronic kidney disorders. As depicted in ROC curves, urine IP-10 andMig demonstrate sensitivity and specificity sufficient to distinguishAR, ABAR, ATN and BKVN from CAN and stable graft function (FIG. 10). Inturn, urine MIP-1δ and OPG demonstrate sensitivity and specificitysufficient to discriminate AR, ABAR, ATN, BKVN, borderline rejection andCAN from stable graft function. While absolute urine levels of IP-10,Mig, MIP-1δ and OPG vary from subject to subject, using 80 pg/ml as acutoff level for each of the markers provides maximal sensitivity andspecificity (FIG. 11). With this threshold, the majority of renaltransplant recipients with AR, ABAR, ATN and BKVN have high levels ofIP-10 and Mig, whereas most of recipients with CAN and stable graftfunction express urine levels of IP-10 and Mig beneath than the cutoff.While MIP-1δ and OPG may overlap with IP-10 and Mig in detection ofacute injuries caused by AR, ABAR, ATN and BKVN, both are stronglypositive in urine samples from recipients with borderline rejection andCAN. To the contrary, the majority of kidney recipients with stablegraft function have a level below the cutoff. One individual in thehealthy controls showed a higher level than the cutoff for IP-10 andMig. Using the 4 biomarkers in aggregate to detect and diagnose kidneyinjuries, including both acute and chronic injuries (AR, ABAR, ATN,BKVN, borderline rejection, and CAN), among the 84 patients that hadrenal allograft injuries only 2 cases (1 AR and 1 ATN) were negative forall the 4 parameters, while for the remaining 82 patients at least 1 ofthe 4 parameters was positive. These results indicate that use of thecurrent panel of the present invention to detect kidney disordersprovides a very high positive detection rate (97.6%).

The diagnostic value of the quadruplex assay of the present inventionwas further evaluated by calculation of indices of sensitivity,specificity, positive predictive value and negative predictive value(FIG. 12). IP-10 and Mig levels are both highly sensitive and highlyspecific to differentiate acute injury (AR, ABAR, ATN and BKVN) fromCAN, stable graft function and healthy renal function. MIP-1δ and OPGare both highly sensitive and highly specific to differentiate acuteinjury (AR, ABAR, ATN and BKVN), borderline rejection and CAN fromstable graft function and healthy renal function.

All publications and patents mentioned in the above specification areherein incorporated by reference. Various modifications and variationsof the described method and system of the invention will be apparent tothose skilled in the art without departing from the scope and spirit ofthe invention. Although the invention has been described in connectionwith specific preferred embodiments, it should be understood that theinvention as claimed should not be unduly limited to such specificembodiments. Indeed, various modifications of the described modes forcarrying out the invention that are obvious to those skilled in therelevant fields are intended to be within the scope of the followingclaims.

1-24. (canceled)
 25. A method of detecting disorders of the kidney,comprising: a) providing; i) a urine sample from a subject, wherein saidsubject is suspected of having a kidney disorder; and ii) reagents fordetection of Mig; and b) detecting the amount of said Mig in said urinesample using said reagents to detect said disorders of the kidney,wherein said amount of said compound in said urine sample is at least 20pg/ml.
 26. The method of claim 1, wherein said amount of said compoundin said urine sample is at least 60 pg/ml.
 27. The method of claim 1,wherein said amount of said compound in said urine sample is at least100 pg/ml.
 28. The method of claim 1, wherein said reagents comprisereagents for performing an immunoassay.
 29. The method of claim 28,wherein said immunoassay is selected from the group consisting of anELISA, radio-immunoassay, automated immunoassay, cytometric bead assay,and immunoprecipitation assay.
 30. The method of claim 1, furthercomprising the step of determining a treatment course of action based onsaid diagnosis of a kidney disorder.
 31. The method of claim 1, furthercomprising the step of determining the presence or absence of aconcurrent infection in said subject.
 32. The method of claim 1, furthercomprising detecting the amount of IP-10 in said urine sample.
 33. Themethod of claim 1, further comprising detecting the amount ofosteoprotogerin in said urine sample.
 34. The method of claim 1, furthercomprising detecting the amount of MIP-1δ in said urine sample.